Fluorescent Tracking Research Articles

Hybrid Decorated Core@Shell Janus Nanoparticles as a Flexible Platform for Targeted Multimodal Molecular Bioimaging of Cancer.

In the recent years, targeted cancer theranosis, the concomitant therapeutic treatment and selective visualization of cancerous tissue, has become a powerful strategy to improve patient prognosis. In this context, targeted multimodal molecular imaging, the combination of different imaging modalities overcoming their individual limitations, has attracted great attention. Due to their unique properties, advanced nanomaterials have taken center stage in the development of theranostics. In this work, we report a novel Janus nanoplatform by combining an Fe3O4 NPs/mesoporous silica core@shell face together with an Au nanoparticle face. Due to its anisotropy, this hybrid nanomaterial enabled the orthogonal site-selective modification of each face permitting the incorporation of a targeting peptide for cancer detection (cRGD) and a fluorescent dye. Due to the intrinsic characteristics of this Janus nanoplatform together with those selectively generated on their surfaces, the resulting hybrid nanocarrier successfully promoted the in vivo tumor-targeted multimodal imaging by magnetic resonance (Fe3O4 core), computed tomography (AuNP face), and fluorescent tracking (fluorescent dye loading) in a fibrosarcoma-bearing mouse model. The achieved results endorse these hybrid Janus nanoparticles as a powerful and flexible platform with integrated imaging and carrier functionalities to be equipped with therapeutic features to generate an advanced multifunctional nanocarrier for targeted cancer theranosis.

GSH-Activated Light-Up Near-Infrared Fluorescent Probe with High Affinity to αvβ3 Integrin for Precise Early Tumor Identification.

The development of tumor-associated, stimuli-driven, turn-on near-infrared (NIR) fluorophores requires urgent attention because of their potential in selective and precise tumor diagnosis. Herein, we describe a NIR fluorescent probe (CyA-cRGD) comprised of a fluorescence reporting unit (a cyanine dye) linked with a GSH-responsive unit (nitroazo aryl ether group) and a tumor-targeting unit (cRGD). The NIR fluorescence of CyA-cRGD with sensitive and selective response to GSH can act as a direct off-on signal reporter for GSH monitoring. Notably, CyA-cRGD possesses improved biocompatibility compared with CyA, which is highly desirable for in vivo fluorescence tracking of cancer. Confocal fluorescence imaging confirmed the tumor-targeting capability and GSH detection ability of CyA-cRGD in tumor cells, normal cells, and coincubated tumor /normal cells and in the three-dimensional multicellular tumor spheroid. Furthermore, it was validated that CyA-cRGD could detect tumor precisely in GSH and integrin αvβ 3 high-expressed tumor-bearing mouse models. Importantly, it was confirmed that CyA-cRGD possessed high efficiency for early-stage tumor imaging in mouse models with tumor cells implanted within 72 h. This method provided significant advances toward more in-depth understanding and exploration of tumor imaging, which may potentially be applied for clinical early tumor diagnosis.

Highly fluorescent, photostable, and biocompatible silicon theranostic nanoprobes against Staphylococcus aureus infections

Human health is severely threatened by bacterial infections, which fuel urgent demand for the development of novel theranostic agents that are suitable for diagnosing and combating bacterial infections. To meet this goal, we synthesized a new kind of theranostic probes, made of vancomycin (Van)-modified fluorescent silicon nanoparticles (SiNPs-Van). The new probes feature good selectivity for Gram-positive bacterial infections, favorable fluorescence, strong resistance to photobleaching (e.g., 12% loss of fluorescence intensity during 40 min continuous laser excitation in the presence of Staphylococcus aureus (S. aureus)), and good biocompatibility (e.g., cell viability > 95% during 24 h treatment). Because of these benefits, the probes were effective at prolonged (i.e., 8-day) fluorescence tracking of S. aureus infections in vivo, thus providing a visible and accurate way to evaluate treatment efficacy. Moreover, the resultant SiNPs-Van with a minimum inhibitory concentration (MIC) of ca. 0.5 μg/mL exhibited high antibacterial efficiency of 92.5%, superior to that (76.5%) of free Van with higher MIC of ca. 1 μg/mL.

Cytotoxicity and autophagy induction by graphene quantum dots with different functional groups.

Graphene quantum dots (GQDs) possess great potential in various applications due to their superior physicochemical properties and wide array of available surface modifications. However, the toxicity of GQDs has not been systematically assessed, thus hindered their further development; especially, the risk of surface modifications of GQDs is largely unknown. In this study, we employed a lung carcinoma A549 cells as the model to investigate the cytotoxicity and autophagy induction of three types GQDs, including cGQDs (COOH-GQDs), hGQDs (OH-GQDs), and aGQDs (NH2-GQDs). The results showed hGQDs was the most toxic, as significant cell death was induced at the concentration of 100 μg/mL, determining by WST-1 assay as well as Annexin-V-FITC/PI apoptosis analysis, whereas cGQDs and aGQDs were non-cytotoxic within the measured concentration. Autophagy detection was performed by TEM examination, LC3 fluorescence tracking, and Western-blot. Both aGQDs and hGQDs induced cellular autophagy to various degrees except for cGQDs. Further analysis on autophagy pathways indicated all GQDs significantly activated p-p38MAPK; p-ERK1/2 was inhibited by aGQDs and hGQDs but activated by cGQDs. p-JNK was inhibited by aGQDs and cGQDs, while activated by hGQDs. Simultaneously, Akt was activated by hGQDs but inhibited by aGQDs. Inhibition of autophagy by 3-MA significantly increased the cytotoxicity of GQDs, suggesting that autophagy played a protective role against the toxicity of GQDs. In conclusion, cGQDs showed excellent biocompatibility and may be considered for biological applications. Autophagy induction may be included in the health risk assessment of GQDs as it reflects the stress status which may eventually lead to diseases.

The Hough Transform as a Basis for Image Recognition and Fluorescent Nanoparticle Tracking

A technique for image processing and the localization (tracking) of fluorescent nanoparticles is developed on the basis of the Hough transform. Features of data analysis in the Hough parametric space that are due to the need to display the coordinates of single point emitters in 3D space are discussed. Examples are given of tracking single point emitters in the form of colloidal semiconductor CdSe/ZnS nanocrystals.

Self-Assembly of Monomeric Hydrophobic Photosensitizers with Short Peptides Forming Photodynamic Nanoparticles with Real-Time Tracking Property and without the Need of Release in Vivo.

Employing nanoscaled materials as photosensitizer (PS) carriers is an effective strategy to solve the problem of poor solubility and low tumor selectivity of hydrophobic PS in photodynamic therapy (PDT), which compulsorily requires the PS release in PDT implementation. However, the complicated environment in vivo makes it difficult to precisely design and control the release process and the delivery process requires real-time tracking. Developing a delivery strategy of hydrophobic PS in the monomeric form with fluorescent emission and without consideration of the PS release in the PDT process, is in urgent demand. Herein, we report a versatile and potent strategy for fabrication of photodynamic nanoparticles (nanoPSs) with featuring the monomeric PS based on aromatic peptide-modulated self-assembly of porphyrin derivatives. Aromatic peptides within nanoPSs can isolate hydrophobic porphyrins from each other, resulting in monomeric porphyrin delivery with real-time fluorescence tracking property and avoiding self-aggregation and hence porphyrin release. Moreover, partially charged porphyrins tend to expose on the surface of nanoPSs, facilitating production and diffusion of 1O2. The highest 1O2 yield can be achieved with porphyrin loading as low as 6 wt %, reducing side effects of excessive porphyrin injection. The nanoPSs show enhanced PDT efficacy in vitro and in vivo leading to complete tumor eradication. This study highlights opportunities for development of active photodynamic nanoparticles and provides an alternative strategy for delivery of hydrophobic photosensitive drugs with enhanced therapeutic effects.

Image-Guided Resection of Glioblastoma and Intracranial Implantation of Therapeutic Stem Cell-seeded Scaffolds.

Glioblastoma (GBM), the most common and aggressive primary brain cancer, carries a life expectancy of 12-15 months. The short life expectancy is due in part to the inability of the current treatment, consisting of surgical resection followed by radiation and chemotherapy, to eliminate invasive tumor foci. Treatment of these foci may be improved with tumoricidal human mesenchymal stem cells (MSCs). MSCs exhibit potent tumor tropism and can be engineered to express therapeutic proteins that kill tumor cells. Advancements in preclinical models indicate that surgical resection induces premature MSC loss and reduces therapeutic efficacy. Efficacy of MSC treatment can be improved by seeding MSCs on a biodegradable poly(lactic acid) (PLA) scaffold. MSC delivery into the surgical resection cavity on a PLA scaffold restores cell retention, persistence, and tumor killing. To study the effects of MSC-seeded PLA implantation on GBM, an accurate preclinical model is needed. Here we provide a preclinical surgical protocol for image-guided tumor resection of GBM in immune-deficient mice followed by MSC-seeded scaffold implantation. MSCs are engineered with lentiviral constructs to constitutively express and secrete therapeutic TNFα-related apoptosis-inducing ligand (TRAIL) as well as green fluorescent protein (GFP) to allow fluorescent tracking. Similarly, the U87 tumor cells are engineered to express mCherry and firefly luciferase, providing dual fluorescent/luminescent tracking. While currently used for investigating stem cell mediated delivery of therapeutics, this protocol could be modified to investigate the impact of surgical resection on other GBM interventions.

Multi-scale pectin/polyphenol beads for non-fouling fluorescence tracking on soft matter under water

The smart polyphenol material could be designed to form two competitive interactions related to same one polyphenol molecule with different objects that could easily adhere to one surface through the weak interaction, and also could be entirely removed from the same surface by the strong interaction. In this study, the multi-scale pectin beads were fabricated by crosslinking with ions that could be acted as polyphenol loading agent through electrostatic force between the ions loading on the surface of pectin beads and polyphenol. The two effects on the size and appearance of pectin beads were detected. Because of the hydrogen bond between polyphenols loading on the beads and the surface of target, fluorescence-functionalized beads could easily adhere on target surfaces. Meanwhile, attributing to stronger electrostatic force between surface ions on beads and polyphenol, the thin membrane made of the beads can be entirely removed from the target surface to avoid the pollution of fluorescence probes.

Immobilization of Photo-Immunoconjugates on Nanoparticles Leads to Enhanced Light-Activated Biological Effects.

The past three decades have witnessed notable advances in establishing photosensitizer-antibody photo-immunoconjugates for photo-immunotherapy and imaging of tumors. Photo-immunotherapy minimizes damage to surrounding healthy tissue when using a cancer-selective photo-immunoconjugate, but requires a threshold intracellular photosensitizer concentration to be effective. Delivery of immunoconjugates to the target cells is often hindered by I) the low photosensitizer-to-antibody ratio of photo-immunoconjugates and II) the limited amount of target molecule presented on the cell surface. Here, a nanoengineering approach is introduced to overcome these obstacles and improve the effectiveness of photo-immunotherapy and imaging. Click chemistry coupling of benzoporphyrin derivative (BPD)-Cetuximab photo-immunoconjugates onto FKR560 dye-containing poly(lactic-co-glycolic acid) nanoparticles markedly enhances intracellular photo-immunoconjugate accumulation and potentiates light-activated photo-immunotoxicity in ovarian cancer and glioblastoma. It is further demonstrated that co-delivery and light activation of BPD and FKR560 allow longitudinal fluorescence tracking of photoimmunoconjugate and nanoparticle in cells. Using xenograft mouse models of epithelial ovarian cancer, intravenous injection of photo-immunoconjugated nanoparticles doubles intratumoral accumulation of photo-immunoconjugates, resulting in an enhanced photoimmunotherapy-mediated tumor volume reduction, compared to "standard" immunoconjugates. This generalizable "carrier effect" phenomenon is attributed to the successful incorporation of photo-immunoconjugates onto a nanoplatform, which modulates immunoconjugate delivery and improves treatment outcomes.

Abstract CT142: Multidrug analyses in cancer patients via intratumoral microdosing with CIVO: A functional approach to precision oncology

Abstract The vision of a precision medicine-guided approach to novel cancer drug development is challenged by high intra-tumor heterogeneity, interpatient diversity, and complex interactions between tumor cells and the surrounding microenvironment. Attempts to model this complexity through the use of ex vivo systems have failed to accurately represent tumor biology, and resulting predictions of clinical drug efficacy have met with limited success. The era of genomics, highlighted by remarkable advances in high-throughput next generation sequencing technology, has sparked hope that this issue can be solved through a precision medicine approach to cancer treatment: matching the right drugs to the right patients. However, despite a few noteworthy successes pairing targeted agents with oncogenic driver mutations, genome-based precision medicine has been limited by an incomplete understanding of the relationship between cancer mutations and drug responsiveness. To overcome these issues, we developed CIVO (Comparative In Vivo Oncology), an arrayed microinjection technology that evaluates tumor responsiveness to microdoses of multiple drugs simultaneously and directly in a patient's tumor. CIVO consists of a multidrug injection device, fluorescent tracking microspheres which accurately denote the position of each drug injection site, and automated image analysis tools capable of quantifying both tumor cell and microenvironmental biomarkers of response to local drug exposure. Here we present preliminary data on the first 4 patients from our clinical feasibility study in soft tissue sarcoma. No unexpected adverse events related to microdose injection were observed. Consistent with historical clinical data, front line agent doxorubicin induced localized increases in markers for DNA damage, tumor cell apoptosis, and macrophage infiltration, whereas gemcitabine did not induce any observable local responses. Doxorubicin also induced tumor cell responses that suggest potential mechanisms of resistance to single agent therapy, including upregulation of PDGFαR and ERK phosphorylation. Multidrug investigation via intratumoral microinjection with CIVO in patients with soft tissue sarcoma appears safe and feasible. The ability to evaluate and cross-compare multiple drugs and drug combinations simultaneously in living tumors across a diverse immune competent patient population provides an innovative and exciting functional approach to complement current precision oncology strategies and enables deep characterization of cancer biology in early drug development. Citation Format: Matthew J. Thompson, Darin J. Davidson, Jessica A. Bertout, Kimberly H. Sottero, Marc O. Grenley, Emily Beirne, Richard A. Klinghoffer, Seth M. Pollack. Multidrug analyses in cancer patients via intratumoral microdosing with CIVO: A functional approach to precision oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT142.

Confined photo-release of nitric oxide with simultaneous two-photon fluorescence tracking in a cellular system

Nitric oxide (NO) is a key signaling molecule in biological systems. New tools are required to therapeutically modulate NO levels with confined precision. This study explores the photoactivatable properties of an NO releasing compound (CPA), based on cupferron O-alkylated with an anthracene derivative. Upon light stimulation, CPA uncages two species: cupferron, which liberates NO, and an anthrylmethyl carbocation, which evolves into a fluorescent reporter. Proof-of-principle is demonstrated using one- and two-photon excitation (1PE and 2PE) in a cellular system (A431 cells). It was found that 1PE induces cell toxicity, while 2PE does not. Since 1PE using UV light is more likely to generate cellular photodamage, the cell toxicity observed using 1PE is most likely a combinatory effect of NO release and other UV-induced damage, which should be subject to further investigation. On the other hand, absence of phototoxicity using 2PE suggests that NO alone is not cytotoxic. This leads to the conclusion that the concept of 2PE photorelease of NO from CPA enable opportunities for biological studies of NO signaling with confined precision of NO release with minimal cytotoxicity.

Detection of neutrons with LiF fluorescent nuclear track detectors

Fluorescent nuclear track detectors (FNTD) based on natLiF crystals were successfully applied for measurements of neutron doses. FNTDs were exposed on a phantom to Hp(10) doses ranging from 1 mSv to 20 mSv. The number of observed tracks was found to be linear with the dose. The actual limit of detection is certainly much lower than 1 mSv and it depends on the scanned volume of a crystal. The very advantageous feature of LiF FNTDs is that the unirradiated detectors show no tracks similar to that produced by thermal neutrons, which means that background (zero-dose signal) is basically equal to zero. These results were obtained with detectors based on natural lithium and might be substantially improved if 6Li enriched lithium is used for crystal growth.It was also found that LiF FNTDs can be used for direct detection (without any converter) not only of thermal, but also of fast neutrons by using recoil of lithium nuclei.

Fluorescent nuclear track detectors for alpha radiation microdosimetry

BackgroundWhile alpha microdosimetry dates back a couple of decades, the effects of localized energy deposition of alpha particles are often still unclear since few comparative studies have been performed. Most modern alpha microdosimetry studies rely for large parts on simulations, which negatively impacts both the simplicity of the calculations and the reliability of the results. A novel microdosimetry method based on the Fluorescent Nuclear Track Detector, a versatile tool that can measure individual alpha particles at sub-micron resolution, yielding accurate energy, fluence and dose rate measurements, was introduced to address these issues.MethodsBoth the detectors and U87 glioblastoma cell cultures were irradiated using an external Am241 alpha source. The alpha particle tracks measured with a Fluorescent Nuclear Track Detector were used together with high resolution 3D cell geometries images to calculate the nucleus dose distribution in the U87 glioblastoma cells. The experimentally obtained microdosimetry parameters were thereafter applied to simulations of 3D U87 cells cultures (spheroids) with various spatial distributions of isotopes to evaluate the effect of the nucleus dose distribution on the expected cell survival.ResultsThe new experimental method showed good agreement with the analytically derived nucleus dose distributions. Small differences (< 5%) in the relative effectiveness were found for isotopes in the cytoplasm and on the cell membrane versus external irradiation, while isotopes located in the nucleus or on the nuclear membrane showed a substantial increase in relative effectiveness (33 – 51%).ConclusionsThe ease-of-use, good accuracy and use of experimentally derived characteristics of the radiation field make this method superior to conventional simulation-based microdosimetry studies. Considering the uncertainties found in alpha radionuclide carriers in-vivo and in-vitro, together with the large contributions from the relative biological effectiveness and the oxygen enhancement ratio, it is expected that only carriers penetrating or surrounding the cell nucleus will substantially benefit from microdosimetry.

Particle tracking by repetitive phase-shift interferometric super resolution microscopy.

Accurate and rapid particle tracking is essential for addressing many research problems in single molecule and cellular biophysics and colloidal soft condensed matter physics. We developed a novel three-dimensional interferometric fluorescent particle tracking approach that does not require any sample scanning. By periodically shifting the interferometer phase, the information stored in the interference pattern of the emitted light allows localizing particles positions with nanometer resolution. This tracking protocol was demonstrated by measuring a known trajectory of a fluorescent bead with sub-5nm axial localization error at 5Hz. The interferometric microscopy was used to track the RecA protein in Bacillus subtilis bacteria to demonstrate its compatibility with biological systems.

The Role of Microenvironment in Preserving the Potency of Adult Porcine Pulmonary Valve Stem Cells In Vitro.

Background and ObjectiveThe potency of tissue resident stem cells is regulated primarily by inputs from the local microenvironment. Isolation of stem cells through enzymatic digestion of tissue may affect epigenetic regulation of cell fate and performance. Here we employ a non-enzymatic method to harvest and investigate tissue resident stem cells from the adult porcine pulmonary valve.Methods and ResultsThe presence of c-Kit+ stem cells within the valve tissue was confirmed by immunohistochemistry. An in vitro culture of minced valve leaflets was developed under the standard conditions (37°C with 5% CO2). The viability of the cellular outgrowths was evaluated over the subsequent 12 weeks. Under this culture condition, we identified a population of non-adherent c-Kit+ cells and multiple cellular structures mimicking the phenotype of embryonic stem cells at different stages of development. Formation of multinucleated cells through cell fusion provided an active niche area for homing and interaction of the non-adherent c-Kit+ cells. Expression of pluripotency markers Oct-4 and Nanog was detected in the newly formed multinucleated cells but not in mature colonies. Partial cell fusion was shown by fluorescent live-cell tracking, which confirmed intercellular molecular exchange between donor and recipient cells, resulting in altered cytoplasmic protein expression by the recipient cell.ConclusionsThese results suggest a role for the microenvironment in decrypting the potential of the valve somatic stem cells in vitro. In addition, our data provide evidence for cell fusion, which may play a critical role in reversing somatic cell fate and spontaneous cellular reprogramming.

Noninvasive photoacoustic and fluorescent tracking of optical dye labeled T cellular activities of diseased sites at new depth.

The migration of immune cells is crucial to the immune response. Visualization of these processes has previously been limited because of the imaging depth. We developed a deep-penetrating, sensitive and high-resolution method to use fast photoacoustic tomography (PAT) to image the dynamic changes of T cells in lymph node and diseases at new depth (up to 9.5 mm). T cells labeled with NIR-797-isothiocyanate, an excellent near-infrared photoacoustic and fluorescent agent, were intravenously injected to the mice. We used fluorescence imaging to determine the location of T cells roughly and photoacoustic imaging is used to observe T-cell responses in diseased sites deeply and carefully. The dynamic changes of T cells in lymph node, acute disease (bacterial infection) and chronic disease (tumor) were observed noninvasively by photoacoustic and fluorescence imaging at different time points. T cells accumulated gradually and reached a maximum at 4 hours and declined afterwards in lymph node and bacterial infection site. At tumor model, T cells immigrated to the tumor with a maximum at 12 hours. Our study can not only provide a new observing method for immune activities tracking, but also enable continuous monitoring for therapeutic interventions.

Cellular-Beacon-Mediated Counting for the Ultrasensitive Detection of Ebola Virus on an Integrated Micromagnetic Platform.

Ebola virus (EBOV) disease is a complex zoonosis that is highly virulent in humans and has caused many deaths. Highly sensitive detection of EBOV is of great importance for early-stage diagnosis for increasing the probability of survival. Herein, we established a cellular-beacon-mediated counting strategy for an ultrasensitive EBOV assay on a micromagnetic platform. The detection platform, which was assisted by on-demand magnetic-field manipulation, showed high integration and enhanced complex-sample pretreatment by magnetophoretic separation and continuous-flow washing. Cellular beacons (i.e., fluorescent cells) with superior optical properties were used as reporters, and each cellular beacon was used as a fluorescent tracking unit to quantify EBOV by counting the numbers of individual fluorescent signals on the micromagnetic platform. This method achieves high sensitivity with a detection limit as low as 2.6 pg/mL, and the detection limit shows little difference in a complex matrix. In addition, it has excellent specificity and good reproducibility. These results indicate that this method proposes an ultrasensitive detection strategy for early diagnosis of the disease.

Theranostic Niosomes for Efficient siRNA/MicroRNA Delivery and Activatable Near-Infrared Fluorescent Tracking of Stem Cells.

RNA interference-mediated gene regulation in stem cells offers great potential in regenerative medicine. In this study, we developed a theranostic platform for efficient delivery of small RNAs [small interfering RNA (siRNA)/microRNA (miRNA)] to human mesenchymal stem cells (hMSCs) to promote differentiation, and meanwhile, to specifically label the transfected cells for the in vivo tracking purpose. We encapsulated indocyanine green (ICG) in a nonionic surfactant vesicle, termed "niosome", that is mainly composed of a nonionic surfactant sorbitan monooleate (Span 80) and a cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). This novel ICG-containing niosome system (iSPN) demonstrated highly efficient siRNA and miRNA delivery in hMSCs. Specific inhibition of miR-138, a negative regulator of osteoblast differentiation, was achieved by iSPN/miR-138, which significantly promoted osteogenesis of hMSCs. Furthermore, iSPN exhibited OFF/ON activatable fluorescence upon cellular internalization, resulting in efficient near-infrared labeling and the capability to dynamically monitor stem cells in mice. In addition, iSPN/siRNA achieved simultaneous long-term cell tracking and in vivo gene silencing after implantation in mice. These results indicate that our theranostic niosomes could represent a promising platform for future development of stem cell-based therapy.

Fluorescent Tracking of Yeast Division Clarifies the Essential Role of Spleen Tyrosine Kinase in the Intracellular Control of Candida glabrata in Macrophages.

Macrophages play a critical role in the elimination of fungal pathogens. They are sensed via cell surface pattern-recognition receptors and are phagocytosed into newly formed organelles called phagosomes. Phagosomes mature through the recruitment of proteins and lysosomes, resulting in addition of proteolytic enzymes and acidification of the microenvironment. Our earlier studies demonstrated an essential role of Dectin-1-dependent activation of spleen tyrosine kinase (Syk) in the maturation of fungal containing phagosomes. The absence of Syk activity interrupted phago-lysosomal fusion resulting in arrest at an early phagosome stage. In this study, we sought to define the contribution of Syk to the control of phagocytosed live Candida glabrata in primary macrophages. To accurately measure intracellular yeast division, we designed a carboxyfluorescein succinimidyl ester (CFSE) yeast division assay in which bright fluorescent parent cells give rise to dim daughter cells. The CFSE-labeling of C. glabrata did not affect the growth rate of the yeast. Following incubation with macrophages, internalized CFSE-labeled C. glabrata were retrieved by cellular lysis, tagged using ConA-647, and the amount of residual CFSE fluorescence was assessed by flow cytometry. C. glabrata remained undivided (CFSE bright) for up to 18 h in co-culture with primary macrophages. Treatment of macrophages with R406, a specific Syk inhibitor, resulted in loss of intracellular control of C. glabrata with initiation of division within 4 h. Delayed Syk inhibition after 8 h was less effective indicating that Syk is critically required at early stages of macrophage–fungal interaction. In conclusion, we demonstrate a new method of tracking division of C. glabrata using CFSE labeling. Our results suggest that early Syk activation is essential for macrophage control of phagocytosed C. glabrata.

Track analysis of the passage of rhodamine-labeled liposomes across porcine jejunal mucus in a microchannel device.

To investigate how surface charge and hydrophilicity affect the mucopermeation of liposomes across intestinal mucus. Rhodamine-labeled liposomes (∼120-130nm) with different surface charges were investigated for their capacity to flux across fresh porcine jejunal mucus in a microchannel device. Fluorescent microscopy and tracking analysis were used to measure liposome movement, while fluorescence lifetime imaging microscopy was utilized to determine mucus pH. Mucopermeation was dependent on hydrophilicity and surface charge- anionic liposomes permeated more than cationic. The most cationic liposomal prototype agglomerated mucus. Presence of Na+, K+ and Mg2+ increased both speed and straightness of the pathways for all prototypes.Cationic but not anionic liposomes caused acidification (pH 2.5). Acidification caused by cationic liposomes explains their ability to interfere with mucus stability. Surface charge of liposomes strongly influences mucopermeation capability.